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WO2002022176A1 - Procede de modulation de neovascularisation - Google Patents

Procede de modulation de neovascularisation Download PDF

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Publication number
WO2002022176A1
WO2002022176A1 PCT/US2001/028954 US0128954W WO0222176A1 WO 2002022176 A1 WO2002022176 A1 WO 2002022176A1 US 0128954 W US0128954 W US 0128954W WO 0222176 A1 WO0222176 A1 WO 0222176A1
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nucleic acid
factor
angiogenesis
neovascularization
acid sequence
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Imre Kovesdi
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Genvec Inc
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Genvec Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1891—Angiogenesic factors; Angiogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/195—Chemokines, e.g. RANTES
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This invention pertains to a method of modulating neovascularization in an animal.
  • Neovascularization or the formation of new blood vessels, is a highly complex and tightly regulated biological process. Neovascularization is normally not continuously required on a large scale in adult animals, thus the process for forming blood vessels is often quiescent except in instances of injury and wound repair. When vascularization is not stringently controlled, serious pathologies can result. For example, reduced vascularization of target tissues is associated with ischemic damage, vascular diseases, necrosis, and muscle wasting. Increased vascularization is associated with, for instance, tumor growth, edema, and diseases of eye, such as diabetic retinopathy and the exudative form of age-related macular degeneration, which are major causes of blindness worldwide.
  • Angiogenic or anti-angiogenic proteins have been administered to an animal to modulate neovascularization.
  • the half-life of some angiogenesis-influencing factors is relatively low, thereby complicating administration of appropriate dosages of peptides to achieve a desired effect.
  • Delivery of an angiogenic or an anti-angiogenic agent to target tissues has also been achieved using gene transfer methods with encouraging results.
  • vascular endothelial growth factor vascular endothelial growth factor
  • delivery of vascular endothelial growth factor has promoted improved perfusion of cardiac tissue and ischemic limbs (see, for example, Isner et al., The Lancet, 348, 370-373 (1996). While perfusion of the target tissue is improved, a more efficient means of modulating neovascularization is needed. In addition, some factors have reduced efficacy under certain conditions. For example, preclinical data suggest that VEGF is less effective in aged individuals. [0004] In view of the above, there exists a need in the art for an alternative and efficient means of modifying vascularization in an animal. The present invention provides such a method. This and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
  • the present invention provides a method of modulating neovascularization in an animal.
  • the method comprises administering to the animal two or more nucleic acid sequences, each nucleic acid sequence encoding at least one angiogenesis-modulation factor that acts upon a different angiogenic process.
  • Each nucleic acid sequence is under the control of separate promoters having different expression profiles.
  • the nucleic acid sequences are expressed to produce the angiogenesis-modulation factors to modulate neovascularization in the animal.
  • the angiogenesis-modulation factor can be an angiogenesis-promoting factor such that neovascularization is induced in the animal.
  • the angiogenesis-modulation factor is an angiogenesis inhibitor and neovascularization is inhibited or reduced in the animal.
  • Figure 1 is a schematic illustrating the homology of various molecular species of vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • Figure 2 is a schematic of VEGF 121 heparin binding variant peptides.
  • the present invention provides a method of modulating neovascularization in an animal.
  • the method comprises administering to the animal two or more nucleic acid sequences, each nucleic acid sequence encoding at least one angiogenesis-modulation factor that acts upon a different angiogenic process, such that the nucleic acid sequences are expressed to produce the angiogenesis-modulation factors to modulate neovascularization in the animal.
  • Modulating neovascularization includes the induction of neovascularization or, in the alternative, the inhibition or reduction of neovascularization.
  • Angiogenesis-modulation factors are gene products that affect the formation of new blood vessels.
  • Angiogenesis-modulation factors can influence the actual generation of new blood vessels, as well as the quality of the blood vessels.
  • the angiogenesis-modulation factors produced by the expression of the nucleic acid sequences act upon different biological processes.
  • the angiogenesis-modulation factors desirably have different mechanisms of modulating neovascularization.
  • Angiogenesis is a complex biological phenomenon that relies on several controlled angiogenic processes. Neovascularization begins with the enzymatic breakdown of the basement membrane of a blood vessel. Endothelial cells migrate to the area of degradation and invade the surrounding extracellular matrix. The invading endothelial cells proliferate to form an elongated column of cells. A lumen then forms within the solid cell column, thereby forming a vessel.
  • Lumen formation involves the differentiation of endothelial cells and the formation of the basement membrane in the new structure.
  • the newly formed vessel structure connects with an existing blood vessel (see, for example, Fotsis et al., J. Nutr., 125: 790S-797S (1995)).
  • the newly formed vessel, as well as existing vessels, also can divide to form branches and capillary networks.
  • the division of existing vessels to form capillary networks is called non-sprouting angiogenesis or intussusception.
  • the above-described processes are dependent on signaling molecules and chemical messengers to regulate and coordinate the biological events such that functional blood vessels are produced.
  • any biological process involved in the formation of new blood vessels e.g., basement membrane breakdown, cell proliferation, cell migration, vessel wall maturation, lumen formation, vessel dilatation, production of mediators, brandling of vessels, etc., is an "angiogenic process.”
  • the angiogenesis-modulating factors modulate different angiogenic processes by acting upon different target molecules.
  • Target molecules refer to, for example, receptors (e.g., growth factor receptors), intracellular signaling molecules, genes, RNA transcripts, and chemical mediators.
  • receptors e.g., growth factor receptors
  • intracellular signaling molecules e.g., genes, RNA transcripts, and chemical mediators.
  • two or more angiogenesis-modulating factors influence neovascularization on multiple fronts, which provides a more complete biological response.
  • each angiogenesis-modulation factor desirably acts upon different cellular signal transduction pathways. Different angiogenic processes rely on different effector molecules and signal transduction pathways for regulation.
  • growth factors such as a vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet derived growth factor (PDGF) act through a tyrosine kinase family receptor system to transmit signals to the cell nucleus.
  • VEGF vascular endothelial growth factor
  • FGF fibroblast growth factor
  • PDGF platelet derived growth factor
  • HTF-l ⁇ and PR39 do not act through tyrosine kinase receptors but, instead, act directly on DNA to promote the production of positive regulators of angiogenesis.
  • HBNF acts by up-regulating the production of urokinase, thereby promoting angiogenesis.
  • soluble growth factor receptors inhibit neovascularization by blocking signaling through growth factor receptors, while pigment epithelium derived factor (PEDF) is a serine protease inhibitor that most likely influences cell-cell or cell-extracellular matrix interactions.
  • PEDF pigment epithelium derived factor
  • Angiostatin works through signal transduction pathways to achieve mitotic arrest.
  • each class of angiogenesis-modulation factor has different mechanisms of action by acting through different target molecules.
  • the present inventive method can be used to induce or promote neovascularization.
  • induction of neovascularization is meant that angiogenesis is either initiated or enhanced. Therefore, for example, when neovascularization is not occurring, the present method provides for initiation of neovascularization. However, if neovascularization is already present, the present method provides a means by which the level of neovascularization is enhanced or heightened.
  • the angiogenesis-modulation factor is an angiogenesis- promoting factor, e.g., a gene product that aids in the formation and/or quality of new blood vessels.
  • a greater degree of neovascularization is induced by the present inventive method compared to neovascularization resulting from administration of one of the nucleic acid sequences alone.
  • the present invention is predicated, in part, on the surprising and unexpected discovery that the use of two nucleic acid sequences, each encoding a different angiogenic-promoting factor that acts upon a different angiogenic process, can induce a greater biological effect than achieved using the maximal levels of angiogenic agents in previously described methods. For example, it was previously believed that the greatest level of angiogenesis possible is achieved by using the maximal dose of, for example, VEGF. However, the present invention provides a method which achieves enhanced neovascularization (an increase in maximal angiogenesis) and a better biological effect.
  • the combination of nucleic acid sequences demonstrates greater than 100% of the angiogenic activity of any one of the nucleic acid sequences (mono therapy) alone.
  • the combination of nucleic acid sequences as used in the present inventive method demonstrates at least 110%, most preferably 120%, of the angiogenic activity of any one of the nucleic acid sequences (monotherapy) alone.
  • Angiogenesis-modulating factors can influence the amount or size of blood vessels formed, or can influence the quality of the newly formed vessels. For instance, instead of increasing the number of new vessels, an angio genesis-promoting factor can promote better conduction through vessels.
  • the angiogenesis-promoting factors used in the present inventive method include, for example, an endothelial mitogen, a factor associated with endothelial migration, a factor associated with vessel wall maturation, a factor associated with vessel wall dilatation, a factor associated with extracellular matrix degradation, or a transcription factor.
  • Endothelial mitogens include, for instance, a vascular endothelial growth factor (VEGF), fibroblast growth factors (FGF, e.g., aFGF, bFGF, and FGF-4), platelet derived growth factor (PDGF), placental growth factor (PLGF), angiogenin, hepatocyte growth factor (HGF), tumor growth factor-beta (TGF-B), connective tissue growth factor (CTGF), and epidermal growth factor (EGF).
  • VEGF vascular endothelial growth factor
  • FGF fibroblast growth factors
  • FGF fibroblast growth factors
  • PDGF platelet derived growth factor
  • PLGF placental growth factor
  • angiogenin hepatocyte growth factor
  • TGF-B tumor growth factor-beta
  • CGF connective tissue growth factor
  • EGF epidermal growth factor
  • Endothelial migration can be induced by, for example, Del-1.
  • Factors associated with vessel wall maturation include, but are not limited to, angiopoietins (Ang, e.g., Ang-1 and Ang-2), tumor necrosis factor-alpha (TNF- ⁇ ), midkine (MK), COUP-TFII, and heparin-binding neurotrophic factor (HBNF, also known as heparin binding growth factor).
  • Vessel wall dilatators include, for example nitric oxide synthase (e.g., eNOS and iNOS) and monocyte chemoattractant protein-1 (MCP-1). Extracellular matrix degradation is promoted by, for instance, Ang-2, TNF- ⁇ , and MK.
  • Suitable transcription factors include, for instance, HLF- l ⁇ and PR39.
  • Other angiogenesis-promoting factors include activin binding protein (ABP) and tissue inhibitor of metalloproteinase (TIMP).
  • Clotting factors such as tissue factor, FVIIa, FXa, thrombin, and activators of PAR1, PAR2, and PAR3 receptors, also are thought to play a role in angiogenesis (see, for example, Carmeliet et al., Science, 293, 1602 (2001)). Additional angiogenic-promoting factors are described in U.S. Patent Application No. 09/832,355.
  • At least one of the nucleic acid sequences encodes a tissue- specific angiogenic factor, most preferably an endothelial-specific angiogenic factor, such as VEGF.
  • angiogenesis-promoting gene product operably linked to suitable expression signals
  • one of the angiogenesis-promoting factors is a VEGF protein or a biologically active (e.g., neovascularization-promoting) fragment thereof.
  • the angiogenic gene product is VEGF 121 , VEGF ⁇ 5 , VEGF 165 , VEGF 18 , VEGF 2 o ⁇ 5 , or a mammalian counterpart, which are variously described in U.S.
  • one of the angiogenesis- promoting factors is VEGF 121 or VEGF ⁇ 65 , particularly VEGF 121 .
  • VEGF 121 does not bind to heparin with a high degree of affinity, as does VEGF 165 .
  • VEGF moieties are advantageous over other angiogenic peptides because VEGF proteins do not induce the growth of tissues not involved in the production of new vasculature.
  • VEGF-II VEGF-C
  • at least one nucleic acid sequence of the present method encodes VEGF or a biologically active fragment thereof and at least one other nucleic acid sequence encodes an angiogenesis-promoting factor other than VEGF or a biologically active fragment thereof.
  • angiogenesis-promoting factors are produced, desirably, a greater degree of neovascularization is induced compared to neovascularization resulting from administration of a nucleic acid sequence encoding VEGF or a biologically active fragment thereof alone.
  • variants, mutants, or chimeras of a VEGF protein can be used in the present inventive method.
  • variants of the VEGF 121 peptide that comprise various segments of the heparin-binding domain of VEGF 189 can be used.
  • VEGF chimeric peptides comprising artificial heparin-binding domains are also appropriate for use in the present inventive method.
  • the heparin-binding domains can be inserted within the angiogenic peptide, or associated with the N- or C- terminus of the peptide, as appropriate.
  • the option of modifying the heparin-binding activity of VEGF peptides provides an addition means of controlling angiogenic activity.
  • the angiogenesis-modulating factor can be an angiogenesis inhibitor such that, when produced, neovascularization is inhibited or reduced in the animal.
  • angiogenesis inhibitor is meant any gene product that prevents or ameliorates neovascularization.
  • complete prevention or amelioration (reduction) of neovascularization is not required in order to obtain a biological effect. Therefore, the present inventive method contemplates both partial and complete prevention and amelioration of angiogenesis.
  • the angiogenesis inhibitors act upon different angiogenic processes. Therefore, angiogenesis inhibitors can, for example, inhibit cell proliferation, cell migration, vessel formation, extracellular matrix degradation, production of mediators, and the like.
  • Angiogenesis inhibitors also can be antagonists for angiogenesis-promoting agents, such that the angiogenesis-promoting factors are neutralized (see, for example, Sato, Proc. Natl. Acad. Sci. USA, 95, 5843-5844 (1998)).
  • Angiogenesis inhibitors suitable for use in the present invention include, for instance, anti-angiogenic factors, cytotoxins, apoptotic factors, anti-sense molecules specific for an angiogenic factor, ribozymes, receptors for an angiogenic factor, an antibody that binds an angiogenic factor, and an antibody that binds a receptor for an angiogenic factor.
  • Anti-angiogenic factors, cytotoxins, and apoptotic factors work to inhibit angiogenesis by directly acting upon cellular functions associated with neovascularization.
  • the anti-angiogenic factors contemplated for use in the present invention include pigment epithelium-derived factor (PEDF), angiostatin, thrombospondin, protamine, vasculostatin, endostatin, platelet factor 4, heparinase, interferons (e.g., LNF ⁇ ), and the like.
  • PEDF pigment epithelium-derived factor
  • angiostatin thrombospondin
  • protamine thrombospondin
  • vasculostatin vasculostatin
  • endostatin endostatin
  • platelet factor 4 heparinase
  • interferons e.g., LNF ⁇
  • active fragments of anti-angiogenic agents i.e., those fragments having biological activity sufficient to inhibit angiogenesis are suitable for use in the present inventive methods.
  • Hyaluronic acid is also known to inhibit endothelial cell proliferation and, therefore, is an appropriate anti-angiogenic agent for use in the present method (Heart Development, Harvey & Rosenthal, eds., Academic Press, New York, 1999, see Chapter 14). Anti-angiogenic agents are further discussed in U.S. Patent 5,840,686; International Patent Applications WO 93/24529 and WO 99/04806; Chader, Cell Different, 20, 209-216 (1987); Dawson et al, Science, 285, 245-248 (1999); and Browder et al, J. Biol. Chem., 275, 1521-1524 (2000).
  • cytotoxins and apoptotic factors include, for example, p53, Fas, Fas ligand, Fas-associating protein with death domain (FADD), caspase- 3, caspase-8 (FLICE), caspase-10, Apo2L, tumor necrosis factor (TNF)-Rl, I ⁇ B, ⁇ Ii B, receptor-interacting protein (RTF)- associated ICH-l/CED-3-homologous protein with a death domain (RATDD), TNF-related apoptosis-inducing ligand (TRAIL), DR4, DR5, a cell death-inducing coding sequence of Bcl-2 which comprises an -terminal deletion, a cell death-inducing coding sequence of Bcl-x which comprises an -terminal deletion, Bax, Bak, Bid, Bad, Bik, Bif-2, c-myc, Ras, Raf, PCK kinase, AKT kinase
  • Apoptotic, cytotoxic, and cytostatic transcription factors can be used in the present method and include, for example, E2F transcription factors and synthetic cell cycle-independent forms thereof, an API transcription factor, an AP2 transcription factor, an SP transcription factor (e.g., an SPl transcription factor), a helix-loop-helix transcription factor, a DP transcription factor (e.g., DPI, DP2, and DP3), and mutants thereof (e.g., dominant negative mutants thereof and dominant positive mutants thereof), and fragments thereof (e.g., active domains thereof), and combinations thereof.
  • E2F transcription factors and synthetic cell cycle-independent forms thereof an API transcription factor
  • an AP2 transcription factor e.g., an SPl transcription factor
  • a helix-loop-helix transcription factor e.g., DPI, DP2, and DP3
  • mutants thereof e.g., dominant negative mutants thereof and dominant positive mutants thereof
  • fragments thereof e.g., active domains
  • Apoptotic, cytotoxic, and cytostatic viral proteins include, for example, an adenoviral El A product, an adenoviral E4/ORF6/7 product, an adenoviral E4/ORF4 product, a cytomegalovirus (CMV) product (e.g., CMV-thymidine kinase (CMV-TK)), a herpes simplex virus (HSV) product (e.g., HSV-TK), a human papillomavirus (HPV) product (e.g., HPVX), and mutants thereof (e.g., dominant negative mutants thereof and dominant positive mutants thereof), and fragments thereof (e.g., active domains thereof), and combinations thereof.
  • CMV CMV-thymidine kinase
  • HSV-TK herpes simplex virus
  • HPV human papillomavirus
  • mutants thereof e.g., dominant negative mutants thereof and dominant positive mutants thereof
  • fragments thereof
  • Cytotoxins and apoptotic factors are particularly useful in inhibiting cell proliferation, an important angiogenic process. Suitable cytotoxins and apoptotic agents can be identified using routine techniques, such as, for instance, cell growth assays and the Tunnel assay, respectively.
  • Anti-sense molecules, ribozymes, soluble receptors, and antibodies to angiogenic factors and receptors can be considered "antagonistic" angiogenesis inhibitors, as they sequester away positive regulators of neovascularization.
  • An anti-sense molecule specific for an angiogenic factor should generally be substantially identical to at least a portion, preferably at least about 20 continuous nucleotides, of the nucleic acid encoding the angiogenic factor to be inhibited, but need not be identical.
  • the anti-sense nucleic acid molecule can be designed such that the inhibitory effect applies to other proteins within a family of genes exhibiting homology or substantial homo logy to the nucleic acid.
  • the introduced anti-sense nucleic acid molecule also need not be full-length relative to either the primary transcription product or fully processed mRNA. Generally, higher homology can be used to compensate for the use of a shorter sequence.
  • Antisense phosphorothiotac oligodeoxynucleotides is exemplary of an anti-sense molecule specific for an angiogenic factor.
  • Ribozymes can be designed that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out tins cleavage, the ribozyme is not itself altered and is, thus, capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature, 334, 585-591 (1988).
  • the ribozyme comprises at least about 20 continuous nucleotides complementary to the target sequence on each side of the active site of the ribozyme.
  • Receptors specific for angiogenic factors inhibit neovascularization by sequestering positive regulators (e.g., growth factors) away from functional receptors capable of promoting a cellular response.
  • soluble VEGF-R1 soluble VEGF-R2 (flk/kdr), and soluble VEGF-R3 (flt-4) receptors, as well as VEGF-receptor- chimeric proteins, compete with VEGF receptors on vascular endothelial cells to inhibit endothelial cell growth (Aiello, PNAS, 92, 10457 (1995)).
  • angiogenic factor-specific antibodies and fragments thereof e.g., Fab, F(ab') 2 , and Fv
  • neutralize angiogenic factors or bind receptors for angiogenic factors e.g., Fab, F(ab') 2 , and Fv
  • At least one nucleic acid sequence encodes a soluble VEGF-R1 receptor and at least one nucleic acid sequence encodes PEDF.
  • VEGF-R1 receptors bind VEGF such that the growth factor is unable to interact with functional receptors to promote endothelial cell proliferation.
  • PEDF is a protease that interferes with cell-cell signaling or the interaction of cells with their environment such that cell migration is blocked.
  • Use of a nucleic acid encoding a soluble receptor in conjunction with a nucleic acid encoding PEDF blocks neovascularization by negatively regulating different angiogenic processes.
  • nucleic acids each encoding at least one angiogenesis-modulation factor, are administered to an animal to modulate neovascularization in the animal.
  • the nucleic acids can comprise DNA, RNA, or a DNA- RNA hybrid, although DNA is preferred.
  • the nucleic acids can be obtained from any source, and can comprise, for example, genomic DNA, cDNA, or synthetic DNA.
  • At least two nucleic acids encoding different angiogenesis-modulation factors are employed in the present method, although more than two nucleic acids (e.g., 3, 4, 5, 6 or more nucleic acids) can be used.
  • the nucleic acid sequences desirably are present on an expression vector suitable for introducing exogenous nucleic acids into host cells.
  • the nucleic acid sequences can be present on separate expression vectors or, in the alternative, can be present on a single expression vector.
  • Suitable expression vectors for use in the present inventive method include, but are not limited to, naked DNA molecules (e.g., plasmids), DNA complexed with transfection- facilitators (e.g., liposomes), and viral vectors, such as adeno-associated virus (AAV)-based vectors, retroviral vectors, herpes simplex virus (HSV)-based vectors, AAV- adenoviral chimeric vectors, and adenovirus-based vectors. Any of these expression vectors can be prepared using standard recombinant DNA techniques described in, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
  • the expression vector of the present inventive method is a viral vector; more preferably, the expression vector is an adenoviral vector.
  • Adenovirus (Ad) is a 36 kb double-stranded DNA virus that efficiently transfers DNA in vivo to a variety of different target cell types.
  • Adenoviral vectors can be produced in high titers and can efficiently transfer DNA to replicating and non-replicating cells, which is advantageous in modulating neovascularization.
  • Newly transferred genetic material remains epi- chromosomal, thus eliminating the risks of random insertional mutagenesis and permanent alteration of the genotype of the target cell.
  • the integrative properties of AAV can be conferred to adenovirus by constructing an AAV- Ad chimeric vector.
  • the AAV ITRs and nucleic acid encoding the Rep protein incorporated into an adenoviral vector enables the adenoviral vector to integrate into a mammalian cell genome. Therefore, AAV- Ad chimeric vectors are an interesting option for use in the present invention.
  • the adenoviral vector can be derived from any serotype of adenovirus.
  • Adenoviral stocks that can be employed as a source of adenovirus can be amplified from the adenoviral serotypes 1 through 51, which are currently available from the American Type Culture Collection (ATCC, Manassis, VA), or from any other serotype of adenovirus available from any other source.
  • ATCC American Type Culture Collection
  • VA Manassis
  • an adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, and 35), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-47), subgroup E (serotype 4), subgroup F (serotypes 40 and 41), or any other adenoviral serotype.
  • an adenovirus is of serotype 2 or 5.
  • Regions of the adenoviral genome can optionally be deleted in order to provide space for insertion of exogenous nucleic acids.
  • regions of the adenoviral genome can be deleted or altered in order to interfere with viral replication.
  • the adenoviral vector used in the present inventive method is preferably deficient in at least one gene function required for viral replication, thereby resulting in a "replication-deficient" adenoviral vector.
  • the adenoviral vector is deficient in at least one essential gene function of the El region (e.g., deficient in at least part of the El a region and/or at least part of the Elb region) of the adenoviral genome, hi addition to a deficiency in the El region, the recombinant adenovirus also can have a mutation in the major late promoter (MLP), as discussed in International Patent Application WO 00/00628. More preferably, the vector is deficient in at least one essential gene function of the El region and at least part of the E3 region.
  • MLP major late promoter
  • the adenoviral vector is "multiply deficient,” meaning that the adenoviral vector is deficient in one or more essential gene functions required for viral replication in each of two or more regions.
  • the aforementioned El -deficient or E1-, E3-deficient adenoviral vectors can be further deficient in at least one essential gene of the E2 region (e.g., the E2a region) and/or the E4 region.
  • Adenoviral vectors deleted of the entire E4 region can elicit lower host immune responses.
  • the adenoviral vector used in the present inventive method can lack all or part of the El region, all or part of the E2 region, all or part of the E3 region, and/or all or part of the E4 region, and any variation thereof.
  • Suitable replication-deficient adenoviral vectors are disclosed in U.S. Patents 5,851,806 and 5,994,106 and International Patent Applications WO 95/34671 and WO 97/21826.
  • an at least E4-deficient adenoviral vector expresses a transgene at high levels for a limited amount of time in vivo and that persistence of expression of a transgene in an at least E4- deficient adenoviral vector can be modulated through the action of a trans-acting factor, such as HSV ICP0.
  • a trans-acting factor such as HSV ICP0.
  • a nucleic acid sequence encoding a trans-acting factor that modulates the persistence of expression of a nucleic acid sequence is preferably provided, whether present on the adenoviral vector or supplied by a second expression vector. The use of trans-acting factors is further described in U.S.
  • Patent 6,225,113 and International Patent Application WO 00/34496 can alter the properties of the viral vector.
  • an adenoviral vector's coat protein can be modified so as to decrease the adenoviral vector's ability or inability to be recognized by a neutralizing antibody directed against the wild-type coat protein, as described in International Patent Application WO 98/40509. Such modifications are useful for long-term treatment of persistent or chronic neovascularization.
  • the coat protein of a viral vector can be manipulated to alter the binding specificity or recognition of a virus for a viral receptor on a potential host cell.
  • adenovirus such manipulations can include deletion of regions of the fiber, penton, pIX, pllla, or hexon, insertions of various native or non-native ligands into portions of the coat protein, and the like.
  • Manipulation of the coat protein can broaden the range of cells infected by a viral vector or enable targeting of a viral vector to a specific cell type.
  • the virus preferably, the adenovirus
  • the viral coat allows the virus to efficiently bind to a broader range of eukaryotic cells than a wild-type virus coat, such as described in International Patent Application WO 97/20051.
  • the viral coat is manipulated such that the virus is "targeted" to a particular cell type, e.g., those cells expressing unique receptors. Suitable modifications to a viral vector, specifically an adenoviral vector, such as, for example, those described herein, are described in U.S.
  • the construction of viral vectors is well understood in the art.
  • Adenoviral vectors can be constructed and/or purified using the methods set forth, for example, in U.S.
  • Adeno-associated viral vectors can be constructed and/or purified using the methods set forth, for example, in U.S. Patent 4,797,368 and Laughlin et al., Gene, 23, 65-73 (1983).
  • the nucleic acid sequences encoding different angiogenic-modulation factors are operably linked to regulatory sequences necessary for expression, i.e., a promoter.
  • a "promoter” is a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis.
  • a nucleic acid sequence is "operably linked" to a promoter when the promoter is capable of directing transcription of that nucleic acid sequence.
  • a promoter can be native or non-native to the nucleic acid sequence to which it is operably linked. Two or more nucleic acid sequences can be under the control of separate regulatory sequences to direct production of the angiogenesis-modulating factors.
  • nucleic acid sequences are present in the same expression vector, the nucleic acid sequences can be under the control of the same regulatory sequences.
  • two nucleic acid sequences can be under the control of a bi-directional promoter.
  • nucleic acid sequences can be arranged as polycistronic sequences, the transcripts of which are processed after transcription.
  • Any promoter i.e., whether isolated from nature or produced by recombinant DNA or synthetic techniques
  • the promoter preferably is capable of directing transcription in a eukaryotic (desirably mammalian) cell.
  • the functioning of the promoter can be altered by the presence of one or more enhancers and/or silencers present on the vector.
  • Enhanccers are cis-acting elements of DNA that stimulate or inhibit transcription of adjacent genes.
  • promoter regions can vary in length and sequence and can further encompass one or more DNA binding sites for sequence-specific DNA binding proteins and/or an enhancer or silencer. Enhancers and/or silencers can similarly be present on a nucleic acid sequence outside of the promoter per se.
  • Suitable viral promoters are known in the art and include, for instance, cytomegalovirus (CMV) promoters, such as the CMV immediate-early promoter, promoters derived from human immunodeficiency virus (HIV), such as the HIV long terminal repeat promoter, Rous sarcoma virus (RSV) promoters, such as the RSV long terminal repeat, mouse mammary tumor virus (MMTV) promoters, HS V promoters, such as the herpes thymidine kinase promoter (Wagner et al, Proc. Natl. Acad.
  • CMV cytomegalovirus
  • HMV human immunodeficiency virus
  • RSV Rous sarcoma virus
  • MMTV mouse mammary tumor virus
  • HS V promoters such as the herpes thymidine kinase promoter (Wagner et al, Proc. Natl. Acad.
  • promoters derived from SV40 or Epstein Barr virus or the Lap 2 promoter
  • promoters derived from SV40 or Epstein Barr virus an adeno- associated viral promoter, such as the p5 promoter
  • an adenoviral promoter such as the Ad2 or Ad5 major late promoter and tripartite leader, and the like.
  • Non- viral promoters for use in the present invention include the regulatory sequences of the metallothionine gene (Brinster et al, Nature, 296, 39-42 (1982)), promoter elements from yeast or other fungi such as the Gal 4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, and the alkaline phosphatase promoter.
  • promoters isolated from the genome of mammalian cells such as the ⁇ -actin promoter, the muscle-creatine promoter, or the elongation factor l ⁇ (EFl ⁇ ) promoter, can be employed.
  • the promoter can be an inducible promoter, i.e., a promoter that is up- and/or down-regulated in response to appropriate signals.
  • suitable inducible promoter systems include, but are not limited to, the hypoxia-driven promoter, the IL-8 promoter, the metallothionine inducible promoter system, the bacterial lacZYA expression system, the tetracycline expression system, and the T7 polymerase system.
  • promoters that are selectively activated at different developmental stages e.g., globin genes are differentially transcribed from globin- associated promoters in embryos and adults can be employed.
  • the regulatory sequences also can comprise a tissue-specific promoter, i.e., a promoter that is preferentially activated in a given tissue and results in expression of a gene product in the tissue where activated.
  • tissue-specific promoter is a myocyte-specific promoter.
  • a promoter exemplary of a myocyte-specific promoter is the myosin light-chain 1 A promoter.
  • a tissue-specific promoter for use in the present inventive vector can be chosen by the ordinarily skilled artisan based upon the target tissue or cell- type.
  • each promoter drives transcription, and, therefore, protein expression, differently with respect to time and amount of protein produced.
  • the CMV promoter is characterized as having peak activity shortly after transduction, i.e., about 24 hours after transduction, then quickly tapering off.
  • the RSV promoter's activity increases gradually, reaching peak activity several days after transduction, and maintains a high level of activity for several weeks.
  • sustained protein expression driven by an RSV promoter in an adenoviral vector is observed in all cell types studied, including, for instance, liver cells, lung cells, spleen cells, diaphragm cells, skeletal muscle cells, and cardiac muscle cells.
  • a promoter, or combination of promoters can be selected for use in the method of the present invention by matching its particular pattern of activity with the desired pattern and level of expression of angiogenesis-modulation factors.
  • the nucleic acid sequences can be under the control of separate promoters having different expression profiles, e.g., at least one nucleic acid sequence is operably linked to an RSV promoter and at least one other nucleic acid sequence is operably linked to a CMV promoter.
  • a hybrid promoter can be constructed which combines the desirable aspects of multiple promoters.
  • the adenoviral vector comprises a nucleic acid sequence encoding a cis-acting factor, wherein the cis-acting factor modulates the expression of the nucleic acid sequences.
  • MAR matrix attachment region
  • immunoglobulin heavy chain ⁇ murine; Jenuwein et al., Nature, 385(16), 269 (1997)
  • locus control region (LCR) sequences or apolipoprotein B (human; Kalos et al., Molec. Cell. Biol., 15(1): 198-207 (1995)), among others.
  • MAR sequences have been characterized as DNA sequences that associate with the nuclear matrix after a combination of nuclease digestion and extraction (Bode et al., Science, 255 (5041), 195-197 (1992)) and augment transcriptional activity of adjacent nucleotide sequences. Similarly, it is believed LCR sequences function to establish and/or maintain domains permissive for transcription. Many LCR sequences give tissue specific expression of associated nucleic acid sequences. Addition of MAR or LCR sequences to the expression vector can further enhance expression of angiogenesis-modulating factors.
  • nucleic acid sequences, and the like, located on an expression vector can be constructed as part of a cassette, either independently or coupled, hi the context of the present invention, a "cassette" is a particular base sequence that possesses functions which facilitate subcloning and recovery of nucleic acid sequences (e.g., one or more restriction sites) or expression (e.g., polyadenylation or splice sites) of particular nucleic acid sequences.
  • nucleic acid sequences With respect to the expression of nucleic acid sequences according to the present invention, the ordinary skilled artisan is aware that different genetic signals and processing events control levels of nucleic acids and proteins/peptides in a cell, such as, for instance, transcription, mRNA translation, and post-transcriptional processing.
  • Protein expression is dependent on the level of RNA transcription that is regulated by DNA signals, and the levels of DNA template.
  • translation of mRNA requires, at the very least, an AUG initiation codon, which is usually located within 10 to 100 nucleotides of the 5' end of the message.
  • the nucleic acid sequence is operatively linked to a polyadenylation site. Also, preferably all the proper transcription signals (and translation signals, where appropriate) are correctly arranged such that the nucleic acid sequence are properly expressed in the cells into which it is introduced.
  • the expression vector also can comprise splice sites (i.e., splice acceptor and splice donor sites) to facilitate mRNA production.
  • the nucleic acid sequence(s) encode a factor that is a processed or secreted protein or acts intracellularly, preferably the nucleic acid sequence further comprises the appropriate sequences for processing, secretion, intracellular localization, and the like.
  • At least one of the angiogenesis-modification factors is linked to an endoplasmic reticular signal peptide, such as that described in Molecular Biology of the Cell, 3 rd Ed., Alberts et al., eds., Garland Publishing, New York (1994).
  • an especially preferred method of modulating expression of a nucleic acid sequence comprises addition of site-specific recombination sites on the expression vector.
  • site-specific recombination to modulate transcription of a nucleic acid sequence is described, for example, U.S. Patents 5,801,030 and 6,063,627 and International Patent Application WO 97/09439.
  • the present inventive method can be used to modulate neovascularization in a variety of tissues, including, for example, skeletal muscle and cardiac tissue. Modulation also can be modulated in adipose tissue (see, for example, U.S. Patent 5,869,037 and International Patent Application WO 97/49827).
  • the choice of expression vector, regulatory sequences, and angiogenesis-modulation factors will depend, in part, on the particular tissue or cell type to be transduced with two or more nucleic acid sequences encoding angiogenesis-modulation factors and the desired biological effect. The ordinarily skilled artisan has the skill to determine the appropriate expression system, regulatory sequences, and angiogenesis-modulation factors for any given tissue or cell-type.
  • the present inventive method can be used for the research or treahent of numerous diseases and asammlungts.
  • the present inventive method of modulating neovascularization can be used to study or treat (therapeutically or prophylactically) coronary artery disease, peripheral vascular disease, congestive heart failure (e.g., left ventricular dysfunction and left ventricular hypertrophy), neuropathy (peripheral or otherwise), avascular necrosis (e.g., bone or dental necrosis), mesenteric ischemia, impotence (or erectile dysfunction), incontinence, arterio-venous fistula, veno-venous fistula, stroke, cerebrovascular ischemia, muscle wasting, pulmonary hypertension, gastrointestinal ulcers, vasculitis, non-healing ischemic ulcers, retinopathies, restenosis, cancer, and radiation-induced tissue injury (such as that common with cancer treatment).
  • congestive heart failure e.g., left ventricular dysfunction and left ventricular hypert
  • the present invention also finds utility in the study and/or aid of wound healing (e.g., healing of ischemic ulcers), plastic surgery procedures (e.g., healing or reattachment of skin and/or muscle flaps), bone healing, ligament and tendon healing, spinal cord healing and protection, prosthetic implant healing, vascular graft patency, and transplant longevity.
  • wound healing e.g., healing of ischemic ulcers
  • plastic surgery procedures e.g., healing or reattachment of skin and/or muscle flaps
  • bone healing, ligament and tendon healing, spinal cord healing and protection e.g., prosthetic implant healing, vascular graft patency, and transplant longevity.
  • angiogenesis-modulation factor delivery of a DNA encoding an angiogenesis-modulation factor can be challenging.
  • the half-life of many angiogenic peptides is very short, the administration of high doses of angiogenic peptides is associated with hypotension, and systemic administration of angiogenic peptides can cause promiscuous induction of angiogenesis in tissues other than that which has been targeted.
  • the quantity of angiogenic peptide delivered is important.
  • the present invention involves the administration of two or more nucleic acid sequences to an animal, each nucleic acid sequence encoding at least one different angiogenesis-modulation factor.
  • an expression vector comprising the nucleic acid sequences are administered in a localized manner, such as accomplished by directly injecting the expression vector into the target tissue or by topically applying the expression vector to the target tissue in which neovascularization is to be modulated.
  • injecting it is meant that the expression vector comprising two or more nucleic acid sequences encoding the angiogenesis-modulation factors is forcefully introduced into the target tissue.
  • Any suitable injection device can be used within the context of the present invention such as that described in, for instance, U.S. Patent 5,846,225. However, it is desirable that whatever means of administering the nucleic acid sequences is chosen, the modulation of neovascularization in non-targeted tissue is minimized.
  • nucleic acid sequences in the treatment of a hindlimb, can be delivered, for example, by intramuscular injection or a catheter inserted into the proximal portion of the femoral artery or arteries.
  • nucleic acid sequences encoding different angiogenesis-modulation factors can be delivered by a catheter or like device inserted sufficiently deeply into the proximal portion of the organ- or tissue-feeding artery or arteries so that gene transfer is effected substantially only into the cells of the target organ or tissue.
  • administration of a dose of the expression vector can be accomplished through a single application (e.g., a single injection or a single topical application) to the target tissue, preferably, administration of the dose is via 2, 3, 4, 5, or more applications (multiple) applications to a target tissue. Preferably, 5 or more applications are administered. More preferably 8 or more applications are administered, and most preferably at least 10 (e.g., 10, 15, 20, 25, 30 or more) applications are administered. Multiple applications provide an advantage over single applications in that they can be manipulated by such parameters as a specific geometry defined by the location on the target tissue where each application is administered. The administration of a single dose of the expression vector via multiple applications can be better controlled, and the effectiveness with which any given dose is administered can be maximized, h this way, too, the undesirable effects associated with administration of a single point application of a large dose can be minimized.
  • a single application e.g., a single injection or a single topical application
  • administration of the dose is via 2, 3, 4, 5, or more applications (multiple
  • the specific geometry of the multiple applications is defined by the location on the target tissue, either in two- or three-dimensional space, where each application of the angiogenic vector is administered.
  • the multiple applications preferably are spaced such that the points of application are separated by up to about 4 cm (e.g., about 0.5-4 cm), more preferably up to about 3 cm (e.g., about 1-3 cm), and most preferably up to about 2 cm (e.g., about 1-2 cm).
  • a single application preferably is administered for up to about 50 cm 3 (e.g., about 0.5-50 cm 3 ) of target tissue, more preferably for up to about 35 cm 3 (e.g., about 1-35 cm 3 ) of target tissue, and most preferably for up to about 15 cm (e.g., about 3-15 cm ) of target tissue.
  • the multiple applications can define any suitable pattern or specific geometry. Therefore, for example, in two- dimensional space, the multiple applications can define a square whereas in three- dimensional space the multiple applications can define a cube.
  • Another parameter of the multiple applications which can be manipulated is the time differential between each application.
  • each of the multiple applications is administered within about 10 minutes (e.g., about 0.5-10 minutes) of each other, more preferably within about 8 minutes (e.g., about 0.5-8 minutes) of each other, and even more preferably within about 6 minutes (e.g., about 1-6 minutes) of each other.
  • all of the multiple applications of the single dose are administered within the aforesaid time frames.
  • each of the multiple applications is administered substantially simultaneously. Administration of multiple applications of angiogenic factors is further described in International Patent Application WO 98/32859.
  • multiple rounds of treatment e.g., 2, 3, 4, 5, or more rounds of treatment
  • a desired biological effect e.g., prolonged enhancement or inhibition of neovascularization
  • Multiple dosing can be facilitated by manipulating the expression vector to evade the immune system.
  • the expression vector preferably evades neutralizing antibodies and other clearance and inflammatory mechanisms.
  • administering can be accomplished either in vivo or ex vivo. Therefore, for example, a target tissue can be removed, contacted with the nucleic acid sequences, and then reimplanted. Ex vivo administration of the angiogenesis-modulators to the target tissue also helps to mimmize undesirable induction of angiogenesis in non-targeted tissue.
  • Any suitable pharmaceutically acceptable carrier can be used within the context of the present invention to administer two or more nucleic acid sequences, and such carriers are well known in the art (see, for example, U.S. Patent 6,225,289 and International Patent Application WO 00/34444).
  • the choice of carrier will be determined, in part, by the particular site to which the composition is to be administered and the particular method used to administer the composition.
  • Formulations suitable for injection include aqueous and non-aqueous solutions, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, immediately prior to use.
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the pharmaceutically acceptable carrier is a buffered saline solution.
  • nucleic acid sequences are administered in small volumes of carrier.
  • Administration of small volumes is such that the tissue in which neovascularization is to be modulated is perfused with the nucleic acid sequences encoding angiogenesis-modulation factors and very little or no nucleic acid sequences encoding the angiogenesis-modulation factors is carried by the blood, lymphatic drainage, or physical mechanisms (e.g. gravitational flow or osmotic flow) to tissues not targeted.
  • the proper dosage of nucleic acids to be administered is such that neovascularization is modulated in the animal.
  • the dosage is sufficient to modulate the level of perfusion of a target tissue. Additionally, the dosage should be such that modulation of neovascularization in non-targeted tissue is minimized.
  • the dosage also will vary depending upon the combination of angiogenesis-modulation factors. Specifically, the dosage will vary depending upon the particular method of administration, including the nature of the vector and nucleic acid sequences encoding and controlling the expression of the angiogenesis-modulation factors. Appropriate dosages can be determined by observing the levels of vascularization and/or perfusion in a tissue using, for example, Doppler imaging to detect blood flow or microscopy to detect changes in vessel number or quality.
  • a dose typically will be at least about lxlO 6 pfu (e.g., Ixl0 6 -lxl0 13 pfu) to a target tissue, for example, a human hindlimb.
  • the dose preferably is at least about lxlO 7 pfu (e.g., about Ixl0 7 -lxl0 13 pfu), more preferably at least about lxlO 8 pfu (e.g., about Ixl0 8 -lxl0 ⁇ pfu), and most preferably at least about lxlO 9 pfu (e.g., about Ixl0 9 -lxl0 10 pfu).
  • the dose typically is for a volume of targeted tissue of about 100 cm , more typically about 150 cm .
  • EXAMPLE 1 This example demonstrates the modulation of neovascularization in the mouse ear model of angiogenesis using the present inventive method.
  • a second adenoviral vector comprising a nucleic acid sequence encoding HBNF (Ad.HBNF), a factor associated with vessel maturation, was co-injected at various dosages (10 10 , 10 9 , 10 8 , and 10 7 pu, n 5).
  • mice All injections were delivered subcutaneously at the base of the ears of anesthetized mice (12 mg/kg xylazine and 60 mg/kg ketamine, IP). Control animals received Ad. VEGF alone. Gross morphological changes to the target tissue were observed at various days post-injection. Serial laser Doppler perfusion measurements were taken at various time points post-injection. Changes in blood vessel number were identified using an Olympus BX40F microscope at 400X to examine harvested ears that had been perfusion fixed and embedded in paraffin.
  • a nucleic acid sequence encoding an endothelial cell mitogen was administered in combination with a nucleic acid sequence encoding a factor involved in vessel maturation to detect modulation of neovascularization in the mouse ear model of angiogenesis described above.
  • Apo E _ " mice were administered adenoviral vectors comprising a nucleic acid sequence encoding VEGF 121 (Ad.VEGF) at a dosage of 10 10 pu.
  • a second adenoviral vector comprising a nucleic acid sequence encoding Ang-1 (Ad.Ang- 1), a factor associated with vessel maturation, was co-injected at various dosages (10 10 , 10 9 , 10 8 , and 10 7 pu, n 5).
  • Perfusion levels were quantified using serial laser Doppler perfusion imaging. At all doses of Ad.Ang-1 administered, except 10 10 pu Ad.Ang-1, peak perfusion was greater for the combination treatment than for administration of Ad.VEGF alone. Increased perfusion induced by the administration of nucleic acid sequences encoding multiple angiogenesis-modulation factors persisted until the end of the trial, Day 13.
  • This example demonstrates a method of modulating neovascularization comprises administering two or more nucleic acid sequences, each encoding one angiogenesis-modulation factor that acts upon a different angiogenic process.
  • neovascularization was induced by administering two nucleic acid sequences encoding different angiogenesis-modulation factors compared to neovascularization resulting from administration of one of the nucleic acid sequences alone.
  • EXAMPLE 2 [0067] This example demonstrates modulation of neovascularization in the rat hindlimb model by administering nucleic acid sequences encoding different angiogenesis-modulation factors with different mechanisms of action.
  • Adenoviral vectors comprising nucleic acid sequences encoding VEGF 121 (Ad. VEGF) or Del-1 (Ad.Del-1) were administered to six-month old male CD rats. The right femoral artery of each rat was removed seven days before administration of the nucleic acids. The rats were divided into five groups. Group 1 was administered Ad.VEGF at 4xl0 10 pu and Ad.Del-1 at 4xl0 9 pu. Group 2 was administered Ad.VEGF at 4xl0 10 pu and Ad.Del-1 at lxlO 9 pu. Group 3 was administered Ad.VEGF at 4xl0 9 pu and Ad.Del-1 at 4xl0 9 pu. The rats of group 4 were administered Ad.
  • Group 5 rats were administered Ad.VEGF alone at a dosage of 4xl0 10 pu.
  • the adenoviral vectors were administered via two injections to the thigh and one injection to the calf of the rat hindlimb.
  • Serial laser Doppler perfusion imaging was used to determine blood flow to foot skin.
  • the rats were sacrificed on Day 28 post-injection for angiography and histological analysis of skeletal muscle to determine capillary and arterial numbers. [0069] At Day 7 post-injection, tissue perfusion levels in Group 2 animals was greater than tissue perfusion levels in Group 5 control animals.
  • tissue perfusion levels in Groups 1, 3, and 4 were all slightly higher than levels detected in Group 5 control animals, i addition, the post-mortem angiogram revealed an approximate 30% increase in angiogram units (blood vessel number) in Group 3 rats compared to the control Group 5 rats.
  • This example demonstrates the ability of the present inventive method to modulate neovascularization in the rat hindlimb model of angiogenesis.
  • a greater degree of neovascularization was induced by administering two nucleic acid sequences encoding different angiogenesis-modulation factors compared to neovascularization resulting from administration of one of the nucleic acid sequences alone.

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Abstract

L'invention concerne un procédé de modulation de néovascularisation chez un animal. Le procédé consiste à administre à l'animal deux ou plusieurs séquences d'acides nucléiques, chaque séquence d'acides nucléiques codant au moins pour un facteur de modulation d'angiogénèse agissant sur différents processus d'angiogénèse, de telle façon que les séquences d'acides nucléiques soient exprimées afin de produire les facteurs de modulation d'angiogénèse modulant la néovascularisation chez l'animal. Moduler la néovascularisation comprend son induction ou, au contraire, sa réduction ou son inhibition.
PCT/US2001/028954 2000-09-15 2001-09-14 Procede de modulation de neovascularisation Ceased WO2002022176A1 (fr)

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